Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.
A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.
Kettling
U
U
Department of Biochemical Kinetics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany. ukettli@gwdg.de
Koltermann
A
A
Schwille
P
P
Eigen
M
M
D001671:Biochemistry / Q000379:methods*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism*; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*
D015246:Deoxyribonuclease EcoRI